Over the past year several issues have been addressed. First, isotopomer analysis coupled with O2 consumption measurements yields absolute flux measurements. This systematic analysis has been reported and is now incorporated in most of our isolated perfused heart studies. Second, we have been interested in analyzing gluconeogenesis and metabolite compartmentation in the liver in vivo. We chose to initiate our studies with [1,2,3-13C] propionate in humans; results of this successful pilot work are described in Research Highlight 2. Third, we have found that the pathway oxaloacetate . PEP . pyruvate . oxaloacetate is undetectable in the heart (as expected) but is very active in the liver. We have found that the 13C NMR spectrum of glucose is quite sensitive to this recycling pathway, and may therefore be used for its quantitation. Fourth, the reliability of the nonsteady-state analysis has been examined. Since the sizes of the combined 6 carbon and 5 carbon pools of the citric acid vary under physiological conditions, if the acetyl-CoA enrichment pattern is shifting rapidly, then the glutamate 13C NMR spectrum may lag and reduce the accuracy of the 13C NMR analysis. This was shown to be a concern if citric acid cycle turnover is slow (K arrest in the heart) or if the acetyl-CoA pool enrichment shifts very rapidly (e.g., 100% [1,2-13C]acetyl-CoA to 100% [2-13C]acetyl-CoA in a few seconds). For physiologically relevant conditions, however, the isotopomer analysis is accurate.